Background: The development of recombinant biologics such as interferon, insulin and Growth Hormone (GH) has had a major impact on the management of diseases including hepatitis, diabetes and GH-deficiency. However, most biologics undergo rapid renal filtration and proteolytic degradation, necessitating frequent dosing regimens. Pegylation can successfully delay clearance but has several drawbacks including potential toxicity, expense and reduced activity. There is therefore need for alternative platform technologies which can generate variable clearance of biological drugs. Hypothesis: That tandems of two molecules joined by a flexible Gly4Ser peptide linker containing a varying number of glycosylation sites, would generate biologics with increased molecular weight (MW) and variable pharmacokinetics.
Methods: We used GH and Leptin (Ob) as our exemplars and cloned, sequenced and expressed hormone-tandems containing up to 4 additional N-linked glycosylations.
Results: Mammalian cell expression was verified by ELISA and western blot. Degree of glycosylation was positively correlated with MW, with addition of 0, 2, 3 & 4 glycans achieving MWs of approximately 47, 56, 63, 68 kDa (GH-tandems) and 40, 50, 54, 58 kDa (Ob-tandems) respectively. Bioactivity was assessed on crude media samples using a transcription luciferase reporter assay (STAT-5 for GH, STAT-3 for Ob). All GH-tandems had appreciable bioactivity regardless of degree of glycosylation: at 0.5 nM GH-tandems with 0 (GHT0) and 4 (GHT5) glycosylations achieved fold-inductions of 6.61 (SE 1.17) and 7.08 (SE 0.19) respectively, compared to rhGH (5.81 (SE 0.18)). Ob-tandems also showed activity comparable to Ob at 0.02 nM. Using Immobilised Metal Affinity Chromatography (IMAC), GH-tandems were purified to homogeneity from suspension adapted serum free cultures (1.9 mg GHT0 from 1L, 1.5 mg GHT5 from 700 ml). Purified rhGh, GHT0 and GHT5 showed comparable bioactivity at 5 nM (fold-inductions; 6.36 (SE 0.30), 6.53 (SE 0.63), 5.65 (SE 0.27) respectively).
Conclusion: Glycosylated-linkers can increase the MW of hormone tandems and provide a potential platform technology for generating long acting biologicals.
Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding: No specific grant from any funding agency in the public, commercial or not-for-profit sector.